Albubody: an engineered scFv variant platform for site-specific drug conjugation and enhanced tumor efficacy

Antibody-drug conjugates (ADCs) represent a promising approach for targeted cancer therapy. Although single-chain variable fragments (scFvs) hold potential as drug carriers, their application in ADCs has been limited. This study introduces Albubody, an engineered scFv fused with an albumin-binding domain (ABD), as a potential ADC scaffold designed to interact with serum albumin in vivo. The human epidermal growth factor receptor 2 (HER2)-targeting Albubody, derived from trastuzumab, is constructed, followed by computational analysis to identify optimal conjugation sites for the cytotoxic drug monomethyl auristatin E (MMAE). The incorporation of the non-natural amino acid p-azido-phenylalanine (pAzF) enables site-specific conjugation using strain-promoted azide-alkyne cycloaddition (SPAAC) chemistry, resulting in the formation of the Albubody-drug conjugate (4D5Albu-MMAE). 4D5Albu-MMAE retained HER2 binding affinity, indicating that site-specific conjugation did not compromise antigen recognition. HER2-specific in vitro cytotoxicity, cellular internalization, and efficient spheroid penetration were also confirmed. Compared to the conventional scFv-drug conjugate (4D5scFv-MMAE), 4D5Albu-MMAE exhibited prolonged systemic exposure and superior antitumor efficacy in HER2-positive xenograft models. This study highlights the potential of Albubody as an effective ADC platform and emphasizes the importance of site-specific conjugation in optimizing therapeutic outcomes for cancer treatment.